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Biomedical subjects

P Talbot

Publications and source records attributed to P Talbot.

At least 55 records · Page 3Linked to original sources
PubMed1990

The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens.

We have expressed a number of polypeptides derived from the capsid proteins of the human parvovirus B19 in Escherichia coli. These include native VP1 (84K) and VP2 (58K) proteins and also fusions to beta-galactosidase containing differing amounts of the amino terminus of the VP1/2 polypeptide. Although each of these was expressed at high levels and the majority were produced as full-length proteins, only one was soluble. This soluble polypeptide, p132, is a beta-galactosidase fusion protein that includes 145 amino acids from B19 which are entirely derived from the region unique to VP1. Despite containing such a small portion of VP1, which itself constitutes only 4% of total capsid protein, p132 reacted with all our known anti-B19 IgM-positive human serum samples. We conclude that this region contains epitopes which must be prominently exposed on the intact virus. We have demonstrated the use of this recombinant antigen in a simple diagnostic assay for B19-specific antibodies which can be used for initial screening of human serum samples. In a survey of 103 serum specimens, our ELISA positively identified all samples (19/19) which were positive by IgM antibody capture radioimmunoassay. The recombinant p132 antigen is efficiently produced and readily purified from E. coli, and its use as a diagnostic antigen should increase the availability of routine clinical testing for human parvovirus infection.

Antigens, Viral↗
PubMed1989

Characterization of the adenovirus proteinase: substrate specificity.

Peptides were synthesized based on the cleavage sites in the adenovirus type 2 proteins pVI and pVII. The synthetic peptides were incubated with disrupted, purified adenovirus as a source of proteinase and specific cleavages were monitored by fast protein liquid chromatography and amino acid analysis. Using this approach it was established that all the peptides cleaved were of the form M(L)XGX decreases G or M(L)XGG decreases X. Thus we have shown that the adenoviral proteinase recognizes a specific secondary structure formed by a sequence of at least five amino acids, the main determinants of specificity being two and four residues to the N-terminal side of the bond cleaved. We were able to examine the relevant structural features of the peptide substrates by utilizing the CHEM-X molecular modelling package. Using our consensus sequence we were able to predict the cleavage sites in the viral proteins pVIII, pre-terminal protein (pTP), 11K and IIIa. Octapeptides containing the predicted sites in pVIII and the pTP were synthesized and shown to be cleaved by the proteinase.

Adenoviruses, Human↗
PubMed1988

A complex cortical reaction leads to formation of the fertilization envelope in the lobster, Homarus.

We have examined the formation of the fertilization envelope in the lobsters Homarus americanus and H gammarus. Oocytes were fixed for electron microscopy either in the ovary or following extrusion from the gonopore. Mature ovarian oocytes are surrounded by a coat (envelope 1), which is comprised of small electron-dense granules and structures resembling "bottlebrushes." At least part of this coat is synthesized by the follicle cells of the ovary. The cortex of ovarian oocytes contains four types of vesicles that we refer to as high-density vesicles (HDV), low-density vesicles (LDV), moderately dense vesicles (MDV), and ring vesicles (RV). Oocytes that were electrically extruded from the gonopore and fixed immediately had an envelope identical to that of ovarian oocytes. The cortex of gonopore oocytes contained the four types of vesicles found in ovarian oocytes. When unfertilized gonopore oocytes were allowed to incubate in sea water, the oocyte cortex appeared unaltered, but envelope 1 swelled and the bottlebrushes dispersed. When recently fertilized oocytes were fixed during natural spawning or following in-vitro fertilization, each type of vesicle was released in sequence from the cortex of the oocyte. The contents of the HDV and LDV appeared first in the perivitelline space, but their fate could not be determined at later times. The ring-shaped elements of the RV and the moderately electron-dense material of the MDV were released exocytotically somewhat later; these materials coalesced in the perivitelline space to form a new coat (envelope 2). Envelope 1 subsequently condensed to its original thickness and appeared firmly attached to envelope 2. Our results show that the fertilized lobster egg is surrounded by two discrete coats. The outer coat, which is formed in the ovary, undergoes a swelling/condensation cycle at spawning. The inner coat originates from a complex cortical reaction. Together these coats comprise the fertilization envelope of the lobster egg.

Animals↗
PubMed1988

Evidence for sequential deployment of secretory enzymes during the normal acrosome reaction of guinea pig sperm in vitro.

Experiments were conducted to determine if acrosomal enzymes are released simultaneously or in sequence during the normal acrosome reaction. Epididymal guinea pig sperm were incubated in a chemically defined, calcium-containing medium which supports normal acrosome reactions within 4-5 hours at 37 degrees C. The sperm suspensions were monitored for motility, normal acrosome reactions, and false acrosome reactions during in vitro incubation. At specified time intervals, the sperm were separated from the incubation medium by centrifugation, and the distribution of dipeptidyl peptidase (DPP II) and acrosin activity was determined by biochemically assaying the hydrolysis of trialanine and N-benzoyl-L-arginine ethyl ester (BAEE), respectively. When calcium was present, there was a significant increase in DPP II activity in the supernatants by 1 hour of incubation and a slight decline at later time points. This release was not correlated with false or normal acrosome reactions (loss of the acrosomal cap) monitored by phase-contrast microscopy but probably represents a very early stage in the normal acrosome reaction. This early stage is difficult to detect at the light microscope level because sperm are still in rouleaux and because membrane fusion is not directly observable. In contrast, acrosin activity, which was assayed in the same supernatants, increased at later times when sperm were observed to have completed normal acrosome reactions. The ultrastructural distribution of DPP II was determined in sperm pellets collected during in vitro incubation by using the DPP II substrate lysyl-alanyl-4-methoxy-2-naphthyamide. In freshly isolated cauda epidiymal sperm, reaction product is confined to the light-staining area in the dorsal bulge of the acrosome. However, by 1 hour of incubation, the light-staining area of many sperm was partially or completely dispersed, while other regions of the acrosome were unchanged. Our data are consistent with the conclusions that DPP II is a highly soluble component of the guinea pig sperm acrosome and that its release occurs during the initial phase of the acrosome reaction while sperm are still in rouleaux. Structural changes in the acrosome associated with DPP II release were detectable by electron microscopy but not by light microscopy. Acrosin, which is less soluble than DPP II, is released at a later time during the acrosome reaction. Both DPP II and acrosin appear to be partially inhibited following their release from sperm. A complete understanding of the sequential release and extracellular activities of the acrosomal enzymes will be necessary to fully define their functions in fertilization.

Acrosin↗
PubMed1987

Plasma immunoreactive atrial natriuretic factor is inhibited by selective blockade of alpha 2-adrenergic receptors in conscious Sprague-Dawley rats.

Effects of alpha 2-adrenergic receptors and calcium channel blockade on basal and clonidine-stimulated immunoreactive atrial natriuretic factor (IR-ANF) in conscious Sprague-Dawley rats were evaluated. Clonidine was injected intravenously (i.v.) in a dose of 50 micrograms. Yohimbine and verapamil were used as a pretreatment, with clonidine in a dose of 50 micrograms and 0.5 mg respectively. The effects of yohimbine (1, 20, 50 micrograms) and verapamil (0.5 mg) on basal IR-ANF were also studied. Plasma IR-ANF was measured by radioimmunoassay with prior extraction on heat-activated Vycor glass. Clonidine injection in a dose of 50 micrograms caused a marked increase of plasma IR-ANF from 34.0 +/- 7.0 pg/ml (mean +/- S.E.M.) to 457.1 +/- 66.3 pg/ml. Clonidine-stimulated ANF secretion was partially inhibited by yohimbine from 457.1 +/- 66.3 pg/ml (mean +/- S.E.M.) to 99.9 +/- 23.1 pg/ml. Moreover, yohimbine in highest doses (50 micrograms) decreased the basal plasma IR-ANF from 34.0 +/- 7.0 pg/ml (means +/- S.E.M.) to 6.8 +/- 3.6 pg/ml. Verapamil did not alter basal and clonidine stimulated IR-ANF. These results indicate the important role played by alpha 2-adrenergic receptors in mediating ANF release.

Animals↗
PubMed1987

Formation of the rupture site in preovulatory hamster and mouse follicles: loss of the surface epithelium.

Changes in the morphology of epithelial cells covering the sides and apex of ovarian follicles were examined in mice and hamsters during the final 13 hr before rupture using light and electron microscopy. At the time of the surge of luteinizing hormone, approximately 13 hr before follicle rupture, epithelial cells along the follicle sides are spherical, covered with microvilli, and remain so throughout the entire cycle. As ovulation approaches, cells at the apex become progressively flatter, increase in diameter, and undergo a reduction in the number and length of microvilli. By 2 hr before ovulation, the microvilli are present only along the boundary between adjacent cells and the cells are in different stages of degeneration. In some cells, the cytoplasm is electron dense and the nuclei are pyknotic. Other cells become electron lucent and cytoplasmic elements are leached from the cell. The apical plasma membrane is lost first over the center of the cell and later over the periphery. Epithelial cells detach from the apex individually until a large patch devoid of cells is formed. This includes the site of eventual rupture. The loss of epithelial cells from the apex of ovarian follicles of other species is compared with our results, and the processes involved in stretching, degeneration, and sloughing of the epithelial cells are discussed.

Animals↗
PubMed1987

Formation of the rupture site in preovulatory hamster follicles: morphological and morphometric analysis of thinning of the granulosa and thecal layers.

The wall of hamster ovarian follicles is composed of the following cell layers: granulosa, theca interna, theca externa, and the surface epithelium. To determine the morphological changes occurring in the follicle during formation of the rupture site, we measured: the thickness of each layer, the number of cells in each layer, and size of cells in each layer, at the apex and base of follicles at specific times during the final 13 hr before ovulation. Changes in the wall occur in 3 stages. During stage 1 (T0-T8), which includes the first 8 hr following the surge in luteinizing hormone, the apical and basal walls thin at the same rate and the antrum increases in size. During stage 2 (T9-T12), there is no change in the thickness of the apical and basal follicle wall nor in the size of the antrum. During the third stage (T12-T13), the size of the antrum decreases slightly and there is an increase in the thickness of the basal wall, which is correlated with its constriction caused by the contraction of smooth muscle cells. The apical wall rapidly thins to the point of rupture. The morphology of cells from each layer is described. Theca interna cells form the final tissue preventing escape of the oocyte-cumulus complex. The roles of cell migration, stretching, and death in thinning of the apical wall are described, and the mechanisms involved in follicle rupture are discussed.

Animals↗
PubMed1987

Mechanism of egg attachment stalk formation in the lobster, Homarus.

We have examined the formation of the egg attachment stalk in the lobsters Homarus americanus and H. gammarus. The formation of the stalk is similar in both species. Ovulated oocytes are surrounded by a single coat, envelope 1, composed of layers 1A and 1B. After passage through the gonopore and exposure to sea water, envelope 1 swells and becomes sticky. A second coat, envelope 2, forms between the oocyte and envelope 1 during a complex cortical reaction initiated after fertilization. Eggs pass over the ventral surface of spawning females to the region of the pleopods, where they stick by means of layer 1A to each other and to the ovigerous setae. Layers 1A and 1B are soft and pliable at this time. During egg attachment, the pleopods beat vigorously and cause envelope 1 to stretch and form attachment stalks. Beating probably also causes the attachment stalks to twist and wrap around the ovigerous setae. After the egg mass is secured to the ovigerous setae, envelope 1 of both the attachment stalk and egg coat condenses to form a tough material capable of securing the egg mass to the pleopods for intervals up to 16 months. After larvae hatch, portions of the egg coats and the attachment stalks are retained on the ovigerous setae until the female undergoes her next molt.

Animals↗
PubMed1987

In vitro penetration of hamster oocyte-cumulus complexes using physiological numbers of sperm.

We have compared the ability of uncapacitated, capacitated acrosome intact, and acrosome-reacted hamster sperm to penetrate the cumulus and corona radiata of fresh hamster oocyte-cumulus complexes (OCC) in vitro. This was done using physiological numbers (1-20) of sperm so that cumulus and corona radiata cells did not disperse during challenge. Uncapacitated sperm did not penetrate to the zona pellucida surface; most (74%) uncapacitated sperm bound to cumulus cells at the periphery of the OCC. Capacitated acrosome-intact sperm penetrated to the zona pellucida surface; a significant percentage of these sperm arrived at the zona pellucida without showing evidence of initiating an acrosome reaction. Most capacitated acrosome-reacted sperm did not enter the extracellular matrix between cumulus and corona radiata cells; those which did penetrated to the zona surface with difficulty, if at all. These results suggest that the changes which occur in the sperm surface during capacitation are more important than the acrosome reaction in enabling hamster sperm to penetrate the cumulus and corona radiata. The effects of gold sodium thiomalate (GST) and polyphloretin phosphate (PPP) (inhibitors of hyaluronidase) on penetration of the OCC by capacitated sperm were also examined. Both synthetic inhibitors blocked sperm penetration to the zona pellucida, but the effective concentrations of inhibitors were far in excess of what was needed to block hyaluronidase activity. Reasons for concluding that the action of these inhibitors is nonspecific are discussed. These data show that hamster sperm with intact acrosomes can penetrate the cumulus and corona radiata cell layers of fresh OCC in vitro and support the hypothesis that the acrosome reaction occurs on the zona pellucida surface.

Acrosome↗
PubMed1985

Sperm penetration through oocyte investments in mammals.

Literature on the interactions between eutherian gametes is reviewed. The oocyte cumulus complex of the female is surrounded by a zona pellucida, corona radiata, and cumulus layer. Sperm undergo an acrosome reaction before penetrating the zona pellucida. The morphological consequences of this reaction and its possible role(s) in penetration of the oocyte cumulus complex are considered. The acrosomal enzyme, hyaluronidase, has been thought to aid sperm in penetrating the cumulus layer and corona radiata. Several recent investigations, including one that shows that motile cells lacking hyaluronidase can penetrate to the zona surface, do not support this idea. Other possible roles of this enzyme in fertilization are discussed. The development of in vitro fertilization systems that employ physiological numbers (1-100) of sperm will be valuable in studying the mechanisms used by sperm to penetrate the oocyte cumulus complex.

Acrosome↗
PubMed1985

Release of hyaluronidase and beta-N-acetylhexosaminidase during in vitro incubation of hamster sperm.

Previous studies have shown that hamster sperm release a significant amount of hyaluronidase before and independently of the normal acrosome reaction. In this study, we have used improved methods for in vitro incubation to investigate the time course of the release of hyaluronidase and hexosaminidase from hamster sperm. When hamster sperm are incubated in medium which allows capacitation, 34 to 47% of the total mechanically extractable hyaluronidase and 34 to 51% of beta-N-acetylhexosaminidase are released into solution prior to and independently of the normal acrosome reaction (ARx). An additional 40 to 50% of the hyaluronidase and 34 to 51% of the hexosaminidase are released at the time of the normal ARx. Control experiments indicate that the early release is not due to the presence of dead sperm in culture and that the normal ARx is required for the second release. Increasing amounts of TCA-precipitated bovine serum albumin in the culture medium stimulated the early (1 hr) release of both enzymes. The data are consistent with the ideas that a significant amount of both enzymes is released from the sperm surface by 1 hr of incubation and that about the same amount of each enzyme is released during the normal ARx. Hyaluronidase and hexosaminidase release at the time of the acrosome reaction was measured for the first time using hamster sperm. The biphasic release of these enzymes may indicate that they have a dual function in fertilization and may help explain how sperm can penetrate the cumulus and corona radiata without undergoing an acrosome reaction.

Acrosome↗
PubMed1985

Motile cells lacking hyaluronidase can penetrate the hamster oocyte cumulus complex.

Sperm hyaluronidase is thought to assist in penetration of the extracellular matrix (ECM) between the cumulus and corona radiata cells surrounding mammalian oocytes. The question was asked: Can motile cells which lack hyaluronidase penetrate the hamster oocyte cumulus complex (OCC)? Sea urchin (Strongylocentrotus purpuratus) and frog (Rana catesbeiana) sperm and the unicellular, biflagellated, green alga Chlamydomonas reinhardtii were extracted and found to contain no hyaluronidase activity. Moreover, none of these cells was able to disperse the cumulus cells of hamster OCC, nor did they affect the ultrastructure of the ECM between cells. Fresh hamster OCC were challenged with suspensions of each cell type. Frog and sea urchin sperm penetrated to the zona pellucida surface in less than 5 min. A cell wall-less mutant of Chlamydomonas also penetrated to the zona surface but required longer than 5 min. Wild-type Chlamydomonas penetrated only halfway to the zona, perhaps because its cell wall adhered to the ECM between the cumulus cells and retarded its movement. The motility of the frog and sea urchin sperm was not affected by the ECM of the OCC. Frog sperm exhibited slow lethargic motility yet had no difficulty penetrating to the zona; this indicates that hyperactivated motility is not required for penetration of the ECM. None of the challenge cells penetrated the zona pellucida, although the frog sperm did compress the weave of the zona. These data show that motile cells which lack the enzyme hyaluronidase can readily penetrate the ECM of the hamster cumulus and corona radiata and suggest that the significance of hyaluronidase in fertilization should be reevaluated.

Animals↗
PubMed1985

Cytochemical localization of dipeptidyl peptidase II (DPP-II) in mature guinea pig sperm.

Guinea pig sperm contain the exopeptidase dipeptidyl peptidase II (DPP-II). Our purpose was to localize this enzyme in sperm at the ultrastructural level using lysylalanyl-4-methoxy-beta-naphthylamide which is the specific synthetic substrate for DPP-II. In substrate incubated sperm, reaction product was located within the acrosome. It was concentrated over the light staining area which forms the prominent dorsal bulge of the acrosome. Within this area there are spherical zones of moderate electron density which had little or no reaction product. Control sperm did not have reaction product. We conclude that DPP-II is restricted to a compartment within the guinea pig sperm acrosome.

Acrosome↗
PubMed1984

Hyaluronidase dissolves a component in the hamster zona pellucida.

Mammalian sperm must pass between cumulus cells and corona radiata cells before reaching the surface of the zona pellucida which surrounds the oocyte. The cumulus and corona radiata cells are separated from each other by an extracellular matrix (ECM) containing hyaluronic acid. The structure of this ECM and of the zona pellucida was investigated in the hamster oocyte-cumulus complex (OCC) using transmission electron microscopy (TEM) following processing in ruthenium red. When fixed in the presence of ruthenium red, the ECM of the OCC and the zona pellucida were well preserved and highly structured. The ECM between corona radiata cells was comprised of a network of granules and filaments which resembled hyaluronic acid containing matrices described in other systems. The outer one-third to one-half of the zona pellucida was porous; the ECM of the corona radiata extended into these pores. Bovine testicular hyaluronidase, Streptomyces hyaluronidase, and hamster sperm extracts containing hyaluronidase each dispersed the cumulus cells and most of the corona radiata cells. TEM examination revealed that brief (5-10 min) hyaluronidase treatment of OCCs removed the matrix filaments and caused clumping of the granules in both the corona radiata and zona pellucida. Longer hyaluronidase treatments (15-30 min) removed both filaments and granules. Our observations are consistent with the ideas that: 1) the ECM between corona radiata cells contains hyaluronic acid, and 2) hyaluronic acid is present in the outer one-third to one-half of the zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Ultrastructure of opossum oocyte investing coats and their sensitivity to trypsin and hyaluronidase.

Ovulated opossum oocytes are surrounded by a zona pellucida, but not by cumulus cells. Opossum sperm carry at least four acrosomal hydrolases (hyaluronidase, acrosin, N-acetylhexosaminidase, and arylsulfatase); the functions of these enzymes in opossum fertilization are uncertain. To identify possible substrates for these hydrolases, the ultrastructure of opossum oocytes was examined after fixation in the presence of ruthenium red which stabilizes extracellular matrices. This oocyte is unusual in having a wide perivitelline space containing a highly structured extracellular matrix (ECM). The ECM is comprised of granules and filaments, and it resembles matrices known to contain hyaluronic acid in other systems. Hydrolases, known to be present in opossum acrosomes, were tested for their effect on the ultrastructure of the zona pellucida and matrix of the perivitelline space. Trypsin dissolved the zona pellucida and decreased the size of the granules in the perivitelline space. Streptomyces hyaluronidase, which specifically attacks hyaluronic acid, removed only matrix filaments. Arylsulfatase, N-acetylhexosaminidase, and beta-glucuronidase did not affect the zona pellucida or ECM in our assay. These observations are consistent with the ideas that (1) opossum sperm must penetrate two oocyte investments, the zona pellucida and ECM of the perivitelline space; (2) the ECM contains hyaluronic acid (filaments) and protein (granules); (3) opossum sperm acrosin may function in penetration of the zona pellucida and ECM; and (4) opossum sperm hyaluronidase may function in penetration of the ECM by degrading hyaluronic acid (filaments). Dissolution of the granules and filaments from oocyte microvilli is probably necessary to permit close apposition and fusion of the sperm and oocyte membranes. The evolutionary significance of these results is discussed.

Animals↗
PubMed1984

A study of efficacy, tolerance and compliance of once-daily versus twice-daily metoprolol (Betaloc) in hypertension. Betaloc Compliance Canadian Cooperative Study Group.

The effects of metoprolol (Betaloc tablets) in a group of 193 hypertensives were compared with the effects of a slow-release formulation (Betaloc Durules) in a further group of 196 patients. Patients were selected at random for treatment. There were no differences between the groups in terms of age, weight, sex, blood pressure, concurrent illness or concomitant therapy. Blood pressure control and apparent adverse effects were similar for both groups; the overall withdrawal rate from each group was similar. Compliance, assessed by tablet counts, was significantly improved in the group receiving once-daily therapy. Simplification of the dosage regimen to once-daily therapy appears to improve the patient's willingness to comply with the physician's instructions.

Clinical Trials as Topic↗